Gene therapy involves the introduction of foreign genes into the cells or tissues of a patient in order to treat hereditary disorders or other diseases such as cancer or AIDS. Early successes with gene therapy have involved the use of the preferred retrovirus-derived vectors to insert genes capable of marking cancer cells, or of treating cancer, or diseases such as severe combined immunodeficiency (reviewed in Anderson, W F, 1992, Science 256:808-813). In early trials with cancer gene therapy by Rosenberg and his colleagues, two patients with advanced metastatic melanoma experienced remission after gene therapy (Rosenberg, S A, 1992, J. Amer. Med. Assoc. 268,2416-2419). However, it is difficult and often impossible to achieve acceptable levels of expression for prolonged periods from such retroviral vectors.
Until recently, gene therapy experiments have taken place only after extensive review, and only a limited number of patients have been treated. A primary reason for such caution stems from the problems associated with the use of retrovirus-derived vectors used to deliver the genes into the cells and chromosomes of the recipient. The most difficult problem has been the ability of the retrovirus-derived vector to genetically recombine with related, retrovirus-derived helper gene sequences present in the donor cell. The combination of the retrovirus vector sequences plus the retrovirus helper sequences together comprise nearly the entire viral genome. When these two parts recombine, the result is an infectious and oncogenic virus which is capable of developing into a full-blown infection, leading to characteristic viremia and cancers in mice and primates. For example, three monkeys undergoing gene therapy trials at The National Institutes of Health died from lymphomas that were subsequently traced to recombination events within the cells used to propagate the virus (Donahue, R E, et al, 1992, J. Exptl. Med. 176, 1125-1135). In addition, retroviral vectors are often transcriptionally silenced after entering the cell. It has been noted that cells of mammals often attach methyl groups to certain regions (called CpG islands) of the viral promoter DNA, apparently preventing the transcription of RNA (Hoeben, R C et al, 1991, J. Virol., 65:904-912). This methylation of CpG residues has been postulated to be primarily a host defense mechanism for eliminating expression from foreign DNA entering the cell, such as a virus. Unfortunately, it also reduces expression from viral vectors used to deliver therapeutic genes, and thus reduces their effectiveness. This problem may be overcome through the use of vectors which are not foreign to cells.
It would be very desirable to invent vectors which have no homology to viral helper sequences, thus preventing the possibility of homologous recombination leading to the production of a replication competent virus. Second, it would be desirable if vectors could be used which have no oncogenic phenotype, or at least a greatly reduced oncogenic phenotype. Thirdly, it would be best if the vectors had a transcriptional promoter with enhanced capability for producing regulated expression in cells. Fourthly, it would be best if the vectors lacked CpG methylation `islands` in their transcriptional promoters, and are normally expressed in living tissue. Finally, it would be best if the vector was made suitable and convenient for human gene therapy, by reducing the load of unnecessary genetic sequences (thus providing more space for foreign therapeutic genes), by including useful cloning and regulatory sites, and by making the vector generally amenable to change, permitting it to be easily adapted for delivery of a wide variety of genetic material. If all these goals could be attained, human gene therapy would be made much safer, and more efficacious. This, in turn, would permit the widespread implementation of gene therapy among afflicted groups of individuals, such as the million or so persons who die of cancer each year in the United States alone. Thus, the implementation of gene therapy as a lifesaving technology will require not one, but several technical improvements over the retroviral vectors currently available.
Limited progress has recently been reported in reaching these theoretical goals. For example, Temin and his colleagues have been successful in combining viruses of different origins in order to decrease homology and homologous recombination in helper cells (U.S. Pat. No. 5,124,263). The oncogenic transcriptional promoter was inactivated by making a deletion at the 3'-end of the virus vector. These changes, combined with the use of safer helper cell lines (U.S. Pat. No. 5,124,236), did decrease the rate of homologous recombination although the resulting viral titers have been disappointing. Others have devised safer helper cell lines in which there is less overlap and homology between the nucleic acid sequences which make the viral genes for transmission (Markowitz et al, 1988, J. Virol. 62, 1120-1124; Markowitz et al, Virology 167, 400-406; WO9205266). Titer is very important since it limits the effectiveness of the viral infection, and the highest titers are attained with retroviral vectors which have a large portion of the gag helper gene sequence intact, thus increasing the level of homologous recombination and RCR. This problem can be partially overcome by introducing multiple mutations in the viral gag gene, however the vectors can still participate in homologous recombination, and they generally have fully-oncogenic transcriptional promoters. The background of retroviral vectorology together with recent advances in patented vectors and public domain technology have recently been reviewed by the applicant (Hodgson, C P, 1993, Curr. Opin. Thera. Patents, 3:223-235.), a copy of which is appended and which shall be referred to in this application, together with other references, as if fully set forth.
Previously, the applicant filed patent applications (pending) covering the Method of Gene Transfer Using Retrotransposons (U.S. Ser. No. 07/603,635, Oct. 25, 1990, and subsequent continuation application; also WO 92/07950), which described the first use of a nonviral mobile genetic element (VL30) for intercellular gene transfer and expression. Previous vectors had used replication-competent or defective viruses derived from a replication-competent oncogenic virus family, thus facilitating recombination and oncogenesis. The vector of choice until now, Moloney murine leukemia virus (MoMLV), is also the vector most commonly used in helper cell lines, including those currently being used in human gene therapy (Miller, A D, and Buttimore, C, 1986, Mol. Cell. Biol. 6:2895-2902; (U.S. Pat. No. 4,861,719). The instant invention describes new retrotransposon VL30 vectors which are made useful for human gene therapy through a number of modifications and improvements.
The use of transposable genetic elements for gene therapy is a natural extension of their evolutionary importance, first described by McClintock (McClintock, B, 1957, Cold Spring Harbor Symp. Quant. Biol. 21:197-216), and represents a fundamental departure from the use of pathogenic agents for gene therapy vectors in the past.
The mobile element VL30 vectors which the applicant described in his previous application were made from a mouse retrotransposon which is present at 100-200 copies in the germ line of mice. The LTR transcriptional units and complete genomes of some of these genes in mice have been characterized by the applicant and others (Hodgson, C P et al, 1983, Mol. Cell. Biol. 3:2221-2231; Adams, S E, et al, 1988, Mol. Cell. Biol. 8:2989-2998; Hodgson, C P, et al 1990, Nucleic Acids Res. 18:673), and transcription from the LTR promoter has been observed by a variety of methods in cell culture, including reporter genes, RNA blotting techniques, etc. (Norton, J D, and Hogan, B L, 1988, Dev. Biol. 125:226-228; Rotman, G, et al, 1986, Nucleic Acids Res. 14:645-658). Comparison of VL30 to other types of transposable elements such as those found in Drosophila (fruitfly) and yeast indicated that they had features in common such as primer binding sites. This suggested that they replicated in a manner similar to retroviruses. But the elements found in great abundance in yeast, mouse, and Drosophila had a remarkable lack of disease phenotype compared to retroviruses. The mouse VL30 genomes sequenced to date have no viral structural genes (see Adams et al, 1988 supra; also Hodgson et al, 1990, supra). Instead, they contain an internal conserved sequence of unknown function, which does not appear to contain any long open reading frames. Unlike MoMLV proviruses, they are abundantly expressed in vivo. In this disclosure, we demonstrate for the first time that a surprising amount of the internal sequences can be removed and replaced with facilitating sequences or additional useful genes which can be delivered by the VL30 and expressed in recipient cells, without negative effects on transcription. This in turn permits more genetic material to be added later. Despite their notable lack of phenotype, VL30 genes are capable of mobilizing in the presence of retrovirus infection, or in retroviral helper cells, using their innate ability to copackage into the viral particle to escape from the cell genome and enter a new host cell during viral transmission. Thus, they provide an additional means for the transmission of nonviral genetic information. This information may then be transmitted to a different location within the same cell, to different cells of the same organism, into the germline, to cells in different organs, or between organisms or between species.